Protein synthesis is performed by the ribosome, which in conjunction with many exogenous factors converts the genetic code into protein. Translation has important practical aspects. The ribosome is a target for many clinically important antibiotics, and tools to monitor the ribosome and translation find use in drug screening. Translation also provides the route from gene to expressed protein. Although laboratory based protein synthesis can be performed, it is a cumbersome process, and improved methods of synthesis, for example to produce peptide drugs, are of interest.
Pharmaceutical drug discovery, a multi-billion dollar industry, involves the identification and validation of therapeutic targets, as well as the identification and optimization of lead compounds. The explosion in numbers of potential new targets and chemical entities resulting from genomics and combinatorial chemistry approaches over the past few years has placed enormous pressure on screening programs. There is a need in drug development to determine the ability of a test compound to affect a potential drug target. It is therefore of great interest to provide a rapid, efficient and inexpensive quantification and screening technique.
The ribosome is the central component of the cellular protein synthesis machinery, which catalyzes peptide bond formation between amino acids attached to transfer RNAs (tRNAs) on adjacent codons of a mRNA. Like all enzymes involved in genetic information transfer, the ribosome must perform protein synthesis in a processive manner at a rapid rate, while maintaining the fidelity of information transfer. As a central process in information transfer, the ribosome and translation is often regulated by external factors and element of the mRNA.
A large body of genetic and biochemical data has established the basic aspects of translation. The 30S subunit interacts with the anticodon portion of the tRNA ligands, whereas the 50S subunit interacts with the 3′ ends of the tRNAs. Ribosomal RNA plays a central role in ribosome function. Conserved portions of ribosomal RNA form the binding sites for the tRNA ligands in the peptidyl tRNA (P site) aminoacyl-tRNA (A site) and the exit site for deacylated tRNA (E site). The peptidyl transferase active site consists solely of RNA. Ribosomal proteins also play a critical role in ribosome function; mutations in ribosomal proteins S12 and S5 cause drug resistance and fidelity phenotypes whereas L7/L12 is critical for factor binding. Protein and RNA work in concert to form the active particle.
The ribosome is the target of a many antibiotics. These small compounds, which include aminoglycosides, tetracyclines, macrolides and chloramphenicols, interfere with distinct steps in translation, including initiation, elongation and termination. Many of the ribosome-directed antibiotics target rRNA, which form the critical functional sites on the ribosome. The antibiotics are thus both powerful mechanistic tools to dissect individual steps of protein synthesis, and lead compounds for the development of novel therapeutic agents. The ribosome and translation are important targets for therapeutic intervention, not only for treatment of infectious disease, but also treatment of human diseases that involve protein expression.
The rich structural information on the ribosome lies in stark contrast to knowledge of its dynamics. Systems that would permit the analysis of translation are of great interest for synthetic and screening methods.
Relevant Publications
The analysis of single molecule fluorescence is disclosed in, for example, Ha et al. (1999) Proc Natl Acad Sci U S A 96(3): 893-8; Ha et al. (1999) Proc Natl Acad Sci U S A 96(16): 9077-82; Weiss (1999) Science 283(5408): 1676-83; and Zhuang et al. (2000) Science 288(5473): 2048-51.
The use of ribosome display is discussed, for example, by Amstutz et al. (2001) Curr Opin Biotechnol 200112(4):400-5; and by Hanes et al. (2000) Methods Enzymol 2000;328:404-30.
Ribosome structure and function are reviewed by Puglisi et al. (2000) Nat Struct Biol 7(10):855-61; and Green and Puglisi (1999) Nat Struct Biol 6(11):999-1003. Eukaryotic ribosome function is reviewed, for example, by Lafontaine et al. (2001) Nat Rev Mol Cell Biol 2(7):514-20.